Biological Influence of the Spacer in NOASA

The defining entity in all NO-NSAIDs is NO. Structure-activity studies with NO-ASA indicated that NO was pivotal for its anticancer effects (Kashfi et al., 2005). However, careful reexamination regarding the contribution to the overall biological effect of each of the three structural components of NO-ASA in which the spacer joining the ASA to the NO-releasing moiety was aromatic, led to the surprising conclusion that the NO-releasing moiety was not required for the observed biological effects. Rather, the spacer was responsible for the biological actions of NO-ASA, with the NO-releasing moiety acting as a leaving group to facilitate the release and activation of the spacer to a quinone methide (QM) intermediate that acted as powerful electrophile such that the ASA component had little or no biological contribution (Dunlap et al., 2007; Hulsman et al., 2007; Kashfi & Rigas, 2007). On this basis, a series of o-, p-, and m-ester-protected hydroxy benzyl phosphates (EHBPs) were synthesized in which the -ONO2 leaving group was replaced by a substituted phosphate and the ASA was replaced by an acetate. Electron-donating/withdrawing groups were also incorporated around the spacer to evaluate their effect on QM formation/stability and biological activity (Kodela, Chattopadhyay, et al., 2008). EHBPs inhibit the growth of various human cancer cell lines, indicating an effect independent of tissue type exercising pleotropic effects involving cell death as well as cell cycle phase transitions and that a QM if formed is influenced by the nature of the substitutes about the benzyl spacer (Kodela, Chattopadhyay, et al., 2008). Transient QMs and related electro-philes if formed during the metabolic activation of NO-ASA can lead to DNA alkylation and (reversible) adduct formation between the QM and deoxyadenosine, deoxyguanosine, deoxycytosine, or thymidine. Using deoxycytosine (dC), which has the potential to form only one (reversible) adduct, the relative reversibility of QM reaction versus bioactivity was determined using a series of EHBPs (Kodela, Rokita, Boring, Crowell, & Kashfi, 2008) that indeed showed a reversible dC adduct was formed, and that electron-donating/withdrawing groups significantly affected the rate of adduct formation/decomposition. As predicted, the m analog did not form a QM a finding consistent with the proposed mechanism of action of NO-ASA (Dunlap et al., 2007; Hulsman et al., 2007; Kashfi & Rigas, 2007).

G. NONO-NSAIDs

The production of NO from nitrate, released from the nitrate esters described above requires a three-electron reduction (Thatcher, Nicolescu, Bennett, & Toader, 2004). However, NONO-NSAIDs do not require redox activation before the release of NO (Velazquez, Praveen Rao, Citro, Keefer, & Knaus, 2007). These agents are based on linking a N-diazen-1-ium-1,2 diolate functional group to a classical NSAID (Fig. 6) yielding compounds

FIGURE 6 The chemical structures of NONO-aspirin. Hybrid ester prodrugs possessing a 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate, A(1) or 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate, A(2), moiety attached via a one-carbon methylene space to the carboxylic acid group of aspirin. In B, the NO-releasing moiety is O -acetoxymethyl 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate. In C, the NO-releasing moiety is O2-acetoxymethyl 1-[(2-hydroxymethyl) pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

FIGURE 6 The chemical structures of NONO-aspirin. Hybrid ester prodrugs possessing a 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate, A(1) or 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate, A(2), moiety attached via a one-carbon methylene space to the carboxylic acid group of aspirin. In B, the NO-releasing moiety is O -acetoxymethyl 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate. In C, the NO-releasing moiety is O2-acetoxymethyl 1-[(2-hydroxymethyl) pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

that are not likely to lead to "nitrate tolerance" (Csont & Ferdinandy, 2005; Fung & Bauer, 1994; Hu Siu, et al., 2007) and also have the potential to generate two equivalents of NO (Velazquez et al., 2007). Another attractive attribute of these classes of NO-releasing compounds is their rich derivatiza-tion chemistry that facilitates the targeting of NO to specific target organ and/or tissue site (Keefer, 2003).

The first agent reported in this compound class had a NONOate (O2-unsubstituted N-diazen-1-ium-1,2-diolate) attached via a one-carbon methylene spacer to the carboxylic acid group of a traditional NSAID (aspirin, ibuprofen, and indomethacin) (Velazquez, Praveen Rao, & Knaus, 2005) (Fig. 6A). In vitro, these agents did not inhibit the enzymatic catalytic activity of COX-1 or COX-2; however, they were equipotent to their traditional NSAID counterparts when evaluated in the carrageenan-induced rat paw anti-inflammatory model. Also, unlike their traditional parent NSAID, these agents had no significant gastric toxicity when given orally (Velazquez et al., 2005). A series NONO-NSAIDS (aspirin, ibuprofen, and indomethacin) was subsequently made that had an O2-acetoxymethyl-1-[N-(2-hydro-xyethyl)-N-methylamino]diazen-1-ium-1,2-diolate moiety as the NO donor (2-HEMA/NO) (Velazquez et al., 2007) (Fig. 6B). Here, the NO-donating moiety was attached via a two-carbon ethyl spacer to the carboxylic acid of the traditional NSAID, and because a secondary dialkyamine was used in their synthesis, the number of possible new NONO-NSAIDs was enormous. Like their predecessors, these agents were nonulcerogenic, and in vitro did not inhibit either COX-1 or COX-2 activity, but showed even better anti-inflammatory properties, suggesting that they were acting as prodrugs, requiring metabolic activation by an esterase to release the parent NSAID. A potential limitation was that hydrolysis would also release one equivalent of the corresponding nitrosoamines that are biologically toxic. To overcome this concern, a second generation of O2-acetoxymethyl-protected (PROLI/NO)-releasing NONO-NSAIDs was developed where a diazeniumdiolate ion obtained from an amine like L-proline was used, the N-nitroso derivative of which is nontoxic (Velazquez, Chen, Citro, Keefer, & Knaus, 2008) (Fig. 6C). These agents were also nonulcerogenic, had better anti-inflammatory properties, and effective analgesic activity. They also produced up to 1.9 mol of NO/mol of compound (Velazquez et al., 2008).

NONO-NSAIDs are an attractive class of compounds; however, there are no data on their chemopreventive potential. Based on the NO-NSAIDs, one might expect these compounds to have potent chemoprevention properties. Recently, the antiulcerogenic, anti-inflammatory, analgesic, and antipyretic effects of an NONO-NSAID were compared directly to that of m-NO-ASA, together with effects on relevant biological markers such as gastric PGE2 and lipid peroxidation levels, superoxide dismutase activity, and TNF-a, levels. In all aspects, the two classes of compounds were similar (K. Kashfi & C. A.Velazquez, unpublished data).

VII. Hydrogen Sulfide-Releasing NSAIDs _

A. Hydrogen Sulfide Signaling

Hydrogen sulfide, H2S, is a colorless gas with a strong odor that until recently was only considered to be a toxic environmental pollutant with little or no physiological significance. However, certain bacteria produce and utilize H2S (Pace, 1997). Mammalian cells also produce H2S, with rat serum having a concentration of ~46 mM (Zhao, Zhang, Lu, & Wang, 2001). H2S in low micromolar levels is also produced in other tissues, for example, brain (Abe & Kimura, 1996; Hosoki, Matsuki, & Kimura, 1997) and vascular tissue (Hosoki et al., 1997; Zhao et al., 2001). However, the highest levels of H2S in the body (low millimolar levels) occur in the lumen of the colon (Magee, Richardson, Hughes, & Cummings, 2000) and are probably due to the nature of the luminal flora. Mitochondria of colonic epithelial cells can utilize H2S as an inorganic substrate (Goubern, Andria-mihaja, Nubel, Blachier, & Bouillaud, 2007).

Most endogenous H2S is produced from L-cysteine by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine p-synthase (CBS) and cystathionine g-lyase (CSE) (Bukovska, Kery, & Kraus, 1994; Erickson, Maxwell, Su, Baumann, & Glode, 1990). The activity of these enzymes is regulated by H2S through negative feedback control. Expression of these enzymes is tissue specific, with some tissues requiring both CBS and CSE for H2S generation, while in others only one of the enzymes is needed (Boehning & Snyder, 2003; Levonen, Lapatto, Saksela, & Raivio, 2000; Lu, O'Dowd, Orrego, & Israel, 1992; Meier, Janosik, Kery, Kraus, & Burkhard, 2001). H2S can also be generated endogenously through none-nzymatic reduction of elemental sulfur (the concentration of which in blood is in the millimolar range (Westley & Westley, 1991)) using reducing equivalents supplied through the glycolytic pathway (Searcy & Lee, 1998). Signs and symptoms of H2S toxicity are well known and are well above those produced endogenously. At concentrations of about 250 ppm, H2S can cause pulmonary edema, and concentrations above 1,000 ppm are lethal. (Beauchamp, Bus, Popp, Boreiko, & Andjelkovich, 1984; Reiffenstein, Hulbert, & Roth, 1992.)

The functional role of H2S at physiologically relevant concentrations in the brain appears to be mediated via activation of ATP-sensitive potassium channels (Wang, 2002). The same appears to be the case in the cardiovascular system since an i.v. bolus of H2S transiently decreased blood pressure of rats, an effect that was mimicked by the KATP channel agonist, pinacidil; and blocked by application of glibenclamide, a KATP channel blocker (Zhao et al., 2001). It is noteworthy that both H2S (Distrutti et al., 2006a) and NSAIDs (Ortiz et al., 2001) appear to exert their analgesic effects via KATP channels. H2S is also a strong reducing agent and may function as an important redox controlling molecule (Kashiba, Kajimura, Goda, & Suematsu, 2002).

B. Anti-Inflammatory Effects of H2S

The role of H2S in inflammation and immunity is controversial, with some studies suggesting an anti-inflammatory effect, whereas others suggest a contribution to immune-mediated tissue injury. In the carragee-nan-induced rat hindpaw model of inflammation, CSE and myeloperox-idase (MPO) activity were increased. Pretreatment with D,L-propargylglycine, a CSE inhibitor, reduced carrageenan-induced hindpaw edema in a dose-dependent manner (Bhatia, Sidhapuriwala, Moochhala, & Moore, 2005). Endotoxin administration to mice increased plasma, liver, and kidney H2S levels that was accompanied by an increase in CSE gene expression in both liver and kidney. There was also histological evidence of lung, liver, and kidney tissue inflammatory damage. Administration of sodium hydrosulfide (an H2S donor) resulted in histological signs of lung inflammation, increased lung and liver MPO activity, and increased plasma TNF-a levels (Li, Bhatia, et al., 2005). Elevated H2S levels occur in the plasma of septic shock patients (Li, Bhatia, et al., 2005). Surprisingly, administration of NO-releasing flurbiprofen to LPS-treated rats resulted in a dose-dependent inhibition of liver H2S synthesis and CSE mRNA levels (Anuar, White-man, Bhatia, & Moore, 2006; Anuar, Whiteman, Siau, et al., 2006). Together, these observations suggest a proinflammatory role for H2S. H2S may also act as a proinflammatory mediator in an animal model of pancreatitis (Bhatia, Wong, et al., 2005; Tamizhselvi, Moore, & Bhatia, 2007).

On the other hand, potent anti-inflammatory effects of H2S have been reported. Administration of carrageenan into a rat air pouch resulted in infiltration of substantial numbers of leukocytes and neutrophils. Pretreat-ment with a number of different H2S donors (NaHS, N-acetylcysteine, and Lawesson's reagent) reduced the number of leukocytes in a dose-dependent manner (Zanardo et al., 2006). This reduction was comparable to that seen by using diclofenac, the NOS inhibitor, L-NAME; or the steroid antiinflammatory, dexamethasone. Pretreatment with p-cyanoalanine, a CSE inhibitor, reversed the inhibitory effects of N-acetylcysteine. Pretreatment with diclofenac, NaHS, or Na2S similarly reduced carrageenan-induced rat paw edema while p-cyanoalanine showed a significant increase in paw swelling in response to carageenan (Zanardo et al., 2006). Through per-oxynitrite scavenging, H2S can inhibit tissue oxidative damage (Whiteman et al., 2004, 2005). H2S has been shown to induce apoptosis in neutrophils (Mariggio et al., 1998), which could contribute to resolution of inflammation (Gilroy, Lawrence, Perretti, & Rossi, 2004). In LPS-stimulated microglia and astrocytes, H2S donors inhibited TNF-a secretion, an anti-inflammatory effect, via inhibition of iNOS and p38 MAPK signaling pathways (Hu, Wong, Moore, & Bian, 2007). In the neuroblastoma cell line SH-SY5Y, H2S inhibited rotenone (a toxin used in vivo and in vitro Parkinson's disease models)-induced cell apoptosis via regulation of p38-and JNK-MAPK pathway (Hu, Lu, Wu, Wong, & Bian, 2009). Thus, it appears that similar to NO, physiological concentrations of H2S produce anti-inflammatory effects, whereas at higher concentrations, which can be produced endogenously in certain circumstances, the effects are proinflam-matory (Li, Bhatia, & Moore, 2006; Wallace, 2007b).

C. H2S Prevents Ulcer Formation Caused by NSAIDs and Promotes Ulcer Healing

Gastric mucosa can also produce H2S that may contribute to its defense against luminal substances. Rats given various NSAIDs (aspirin, indomethacin, ketoprofen, or diclofenac), NaHS, or the combination of an NSAID plus NaHS were evaluated for gastric damage, effects on H2S-synthesizing enzymes, gastric blood flow, and other parameters relevant to tissue injury (Fiorucci et al., 2005). NaHS significantly inhibited gastric mucosal injury, TNF-a, ICAM-1, and lymphocyte function-associated antigen-1 mRNA upregulation induced by aspirin. NaHS prevented the associated reduction of gastric mucosal blood flow and reduced ASA-induced leukocyte adherence in mesenteric venules. It did not however, alter suppression of PGE2 synthesis by NSAIDs. Glibenclamide (KATP channel antagonist) and D,L-propargylglycine (CSE inhibitor) exacerbated, whereas pinacidil (KATP channel agonist) attenuated gastric injury caused by ASA. Exposure to NSAIDs reduced H2S formation and mRNA and protein expression of CSE (Fiorucci et al., 2005). These results suggested that suppression of mucosal H2S synthesis may represent another mechanism, in addition to inhibition of COX activity, through which NSAIDs produce GI damage (Wallace, 2007a).

To evaluate the ulcer healing properties of any GI-sparing agent, a chronic ulcer model is generally employed with acetic acid being used to produce gastric ulcers in rats, which highly resemble human ulcers in terms of pathological features and healing mechanisms (Okabe & Amagase, 2005). These ulcers respond well to antiulcer drugs such as the proton pump inhibitors and sucralfate (Okabe & Amagase, 2005). In this model, induction of gastric ulceration was associated with a increase in expression of CSE and CBS enzymes as well has H2S levels, suggesting a defensive response (Wallace, Dicay, McKnight, & Martin, 2007). Ulcer healing was observed following administration of three chemically distinct (Lawesson's reagent, 4-hydroxyhiobenzamide, and H2S-5-ASA) H2S donors, and administration L-cysteine, a precursor of endogenous H2S synthesis, also enhanced ulcer healing (Wallace, Dicay, et al., 2007). In evaluating the possible mechanisms through which H2S accelerated ulcer healing, it was determined that the H2S donors did not raise gastric pH or inhibit acid secretion; KATP channel agonists or antagonists had no affect on ulcer healing; ulcer healing was NO independent; and L-cysteine did not affect gastric levels of glutathione. Since H2S is a vasodilator, it is possible that this may have contributed to healing process observed in this study (Wallace, Dicay, et al., 2007).

D. Hydrogen Sulfide-Releasing NSAIDs for Treatment of Inflammatory Diseases

Research in the field of hydrogen sulfide-releasing NSAIDs (HS-NSAIDs, Fig. 7) is in its infancy. To date, there have been no reports describing the effects of HS-NSAIDs in any in vitro studies of human cancer cell lines or in any in vivo animal models of cancer. There are some studies focusing on HS-diclofenac, HS-indomethacin, and HS-mesalamine for treatment and prevention of inflammatory bowl disease (IBD; Crohn's disease and ulcerative colitis), also their anti-inflammatory properties, and GI-sparing effects have been described.

IBD is a chronic disorder characterized by extensive ulceration and inflammation. The first-line therapy for mild-to-moderate IBD is up to 6g/day of mesalamine (5-aminosalicylic acid), the mechanism of action of which is not well understood (Hanauer, 2006). The animal model used for evaluating various agents for treatment of IBD makes use of trinitrobenzene sulfonic acid (TNBS). This model is well characterized, and exhibits responsiveness to various therapies similar to those used for human IBD and shares many features with IBD in humans, particularly Crohn's colitis (Fiorucci et al., 2002; Morris et al., 1989). Using this model in mice, HS-mesalamine was more effective than mesalamine in reducing mucosal injury and disease activity (body weight loss, fecal blood, and diarrhea) and colonic granulocyte infiltration (Fiorucci et al., 2007). Treatment with mesalamine did not affect the expression of any of several proinflammatory cytokines/chemokines studied. TNBS colitis, like Crohn's colitis, is generally regarded as being driven by Th1 cytokines, including IL-1, IL-2 TNF-a, IFN-g, and IL-12 (Sartor, 1997; Stallmach et al., 1999). The chemokine, RANTES, has also been implicated in the pathogenesis of colitis in this model (Ajuebor, Hogaboam, Kunkel, Proudfoot, & Wallace, 2001). Treatment with HS-mesalamine reduced the expression of mRNA for TNF-a, IFN-g, IL-1, IL-2, IL-12 p40, and RANTES (Fiorucci et al., 2007). Using the same model in rats, HS-mesalamine modulated expression of colonic proin-flammatory mediators, COX-2 and IL-1p (Distrutti et al., 2006b).

Anti-Inflammatory Agents as Cancer Therapeutics Traditional NSAID H2S-releasing moiety

Anti-Inflammatory Agents as Cancer Therapeutics Traditional NSAID H2S-releasing moiety

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